Our goal in this study is to discovery of diagnostic and prognostic urinary non- invasive biomarkers for acute rejection in pediatric kidney transplantation. Graft survival after kidney transplantation, the treatment of choice for children with end stage kidney disease, is negatively impacted by graft injury from acute rejection (AR), and incomplete recovery of acute rejection results in chronic rejection and subsequent early graft loss. Specific and sensitive biomarker discovery for diagnosis of the onset of AR and for prediction of treatment stratification (steroids vs antibody), would be vital for improving the success rate of graft survival. In the absence of an effective, non-invasive test to diagnose acute rejection, the invasive transplant biopsy is the only gold standard, where AR severity and treatment response is difficult to predict. Because of aforementioned reasons, there is a clinical urgency to identify a more specific for a better diagnostic and predictive tool for AR, which could eventually replace the protocol biopsy. The advancement in high throughput techniques such as genomics and proteomics is considered as major step forward in understanding of complex bimolecular processes and biomarker discovery. Urine represents a filtrate of plasma, and is, therefore, an ideal body fluid of study due to the non-invasive nature of the collection process. Urine is a particularly ideal biologic fluid source of study in renal transplantation because it may be reflective of both local processes within the kidney as well as a reflection of changes within plasma. Our proposal is to use highly sophisticated liquid chromatography mass spectrometry (LCMS) methods which has already been proven to be capable of identifying thousands of proteins and relative quantification of the individual proteins present in bio fluids such as plasma and urine as well as label-free LC-MALDI, which identifies urinary peptide signatures. Although, these methods may not be directly applicable to the clinical setting, the information gained from our hypothesis generation stage in Aim 1, is easily transferred to a more rapid, higher- throughput method such as quantitative mass spectrometry (MS) in Aim2, which is ideal for clinical diagnostics. Which eventually will be vital for discovery for diagnosis and prediction of AR treatment stratification thereby improving patient clinical monitoring and transplant survival. PUBLIC HEALTH RELEVANCE: Acute rejection of transplanted kidney still remains the issue to be addressed effectively. In the absence of an effective noninvasive way of diagnosis, kidney biopsy is the only way to monitor the clinical progress the transplanted kidney. In this study, we propose to utilize two most powerful proteomic techniques to identify non-invasive proteomic biomarkers for acute rejection.